This was the first year for this project, which will use biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimer's disease, and excitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Toward this end, we have successfully expressed and purified a prokaryotic glutamate transporter, which is a close cousin of the mammalian protein. Using the bacterial protein allows us to analyze parts of the molecule which would be difficult or impossible to assess with current mammalian expression systems. We have demonstrated that the expressed transporters are functional. We will use cysteine-scanning mutagenesis and site-directed fluorescence labeling to determine which parts of the molecule move during the transport process and characterize these movements in detail.